Basement membranes (BMs) are present in almost all tissues. Throughout the entire life cycle they are decisive for tissue development, maintenance, and repair. Moreover, they are important mechanical barriers against tumor cell invasion and metastasis. At the same time the porous BM protein scaffolds function as growth factor reservoirs and affect the diffusion of biomacromolecules which influences cell differentiation and final tissue function. Despite the paramount importance of BMs, their material properties are still insufficiently understood. Here, we used a non-tumorigenic epithelial cell line (MCF10A) originally isolated from human breast gland tissue. Upon cultivation in Engelbreth Holm Swarm (EHS) hydrogel (3D culture), these cells form hollow, multi-cellular spheroids enclosed by an endogeneously formed BM structure. We separated such BM covered MCF10A acini from the EHS matrix and imaged their structures with scanning force microscopy (AFM), scanning electron microscopy and high-resolution confocal fluorescence microscopy (Airyscan super-resolution technology). Moreover, the resistance against indentation of intact BM shells was tested at different developmental stages. Beyond these structural investigations we exposed BM shells to fluorescent dextran solutions and observed delayed permeation for larger molecular weights and fully matured spheres.Taken together, we used MCF10A derived acini as model systems for physiological basement membranes and characterized their structure, mechanics and permeability for macromolecules. This model system enables clear-cut experiments towards a better understanding of healthy breast gland tissue function and mechanobiological processes in breast cancer invasion.